Abstract Background In the ART-PRO pilot trial there were no virological failures through 48 weeks of treatment with dolutegravir plus lamivudine in suppressed individuals with and without archived lamivudine resistance-associated mutations (RAMs) detected through next-generation sequencing (NGS) but without evidence of lamivudine RAMs in baseline proviral DNA population sequencing. Objectives To present 96 week results from ART-PRO. Methods Open-label, single-arm pilot trial. At baseline, all participants switched to dolutegravir plus lamivudine. Participants were excluded if proviral DNA population genotyping detected lamivudine RAMs. To detect resistance minority variants, proviral DNA NGS was retrospectively performed from baseline samples. For this analysis the efficacy endpoint was the proportion of participants with <50 HIV-1 RNA copies/mL at week 96. Safety and tolerability outcomes were incidence of adverse events and treatment discontinuations. Results Forty-one participants were included, 21 with lamivudine RAMs in historical plasma RNA genotypes. Baseline proviral DNA NGS detected lamivudine RAMs (M184V/I and/or K65R/E/N) above a 5% threshold in 71.4% (15/21) and 15% (3/20) of participants with and without history of lamivudine resistance, respectively. At 96 weeks, 90.2% of participants achieved the efficacy endpoint. Between week 48 and 96 there was one discontinuation due to consent withdrawal and no discontinuations related to adverse events. Two participants had a transient viral rebound, both re-suppressed on dolutegravir plus lamivudine. Through week 96, there were no virological failures. Conclusions In this pilot trial, dolutegravir plus lamivudine maintained virological suppression at 96 weeks despite historical lamivudine resistance and persisting archived minority lamivudine RAMs.